AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriop...AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.展开更多
基金Supported by The NIH-AREA Grant,No.1R15GM52696-01the NSF-RUI Grant,No.DMB-8608480+2 种基金the Howard Hughes Medical Research Institute(two grants to the PLNU Biology Department)Research Associates of PLNU(a 300 member alumni support group)the PLNU Administration
文摘AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.
